Received November 2, 2017; Revised February 19, 2018; Accepted March 7, 2018.

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Abstract

Plant growth promoting rhizobacteria and endophytic bacteria were isolated from different varieties of turmeric (Curcuma longa L.) from South India. Totally 50 strains representing, 30 PGPR and 20 endophytic bacteria were identified based on biochemical assays and 16S rDNA sequence analysis. The isolates were screened for antagonistic activity against Pythium aphanidermatum (Edson) Fitzp., and Rhizoctonia solani Kuhn., causing rhizome rot and leaf blight diseases in turmeric, by dual culture and liquid culture assays. Results revealed that only five isolates of PGPR and four endophytic bacteria showed more than 70% suppression of test pathogens in both assays. The SEM studies of interaction zone showed significant ultrastructural changes of the hyphae like shriveling, breakage and desication of the pathogens by PGPR B. cereus (RBac-DOB-S24) and endophyte P. aeruginosa (BacDOB-E19). Selected isolates showed multiple Plant growth promoting traits. The rhizome bacterization followed by soil application of B. cereus (RBacDOB-S24) showed lowest Percent Disease Incidence (PDI) of rhizome rot and leaf blight, 16.4% and 15.5% respectively. Similarly, P. aeruginosa (BacDOB-E19) recorded PDI of rhizome rot (17.5%) and leaf blight (17.7%). The treatment of these promising isolates exhibited significant increase in plant height and fresh rhizome yield/plant in comparison with untreated control under greenhouse condition. Thereby, these isolates can be exploited as a potential biocontrol agent for suppressing rhizome rot and leaf blight diseases in turmeric.

Turmeric (Curcuma longa L., Family-Zingeberaceae) is a rhizomatous perennial herb cultivated in Indian sub-continent and middle East countries. Dried rhizomes are used as condiment, dye, drug and for cosmetics. India is the leading producer of turmeric in the world and contributes about 75–80% of the world production followed by China, Myanmar, Nigeria and Bangladesh. The main turmeric producing states in India are Andhra Pradesh, Tamil Nadu, Orissa, West Bengal, Maharashtra, Karnataka and Kerala (Thiripurasundari and Selvarani, 2014). The fungus P. aphanidermatum and R. solani cause rhizome rot and leaf blight diseases in turmeric plants and reduce commercial value (Park, 1934; Roy, 1992). It has been noted that chemical fungicides like Ridomil, Metalaxyl, Carbendazim (0.1%) and Mancozeb (0.25%) were commonly used to manage rhizome rot and leaf blight diseases (Muthukumar et al., 2011; Rathaiah, 1982). Use of chemical fungicides is of public concern as it causes various human health problems and also pathogens build resisitance against fungicides. The need for adopting environment friendly disease control measures such as biological control strategies are emphasized presently (Hallmann et al., 2009). An alternative to chemical fungicides for the management of plant diseases is the use of soil borne, non-pathogenic rhizospheric or endophytic bacteria.

Endophytes are ubiquitous and have been found in all species of plants. In general, Endophytes could produce different plant hormones to enhance the growth of the host plants (Waqas et al., 2012). Bacterial endophytes colonize the internal tissues of the plant showing no negative effect on their host (Schulz et al., 2006). In comparison to PGPR, endophytes showed better adaptations against biotic and abiotic stresses, that leads to enhanced plant growth (Pillay and Nowak, 1997). Many endophytes constitute the common rhizospheric bacteria (Burkholderia, Pseudomonas and Bacillus) that produce various secondary metabolites, volatile compounds and antibiotics to counter the deleterious effect of pathogens through mechanisms similar to that of PGPR (Lodewyckx et al., 2002).

Several reports are available on the isolation of PGPR and endophytes, and their effects on growth and yield of crops. The biocontrol agents like T. viride, P. chlororaphis and B. subtilis were used for suppression of rhizome rot of turmeric (Kavitha et al., 2012; Ramarethinam and Rajagopal, 1999). Nevertheless, very little information is available on the effect of native multi trait PGPR and endophyes on growth promotion and biocontrol of rhizome and leaf blight diseases of turmeric. This study was taken up to profile the rhizobacteria and endophytic bacteria associated with turmeric and to evaluate their antagonistic activities, biocontrol potential and plant growth promotion both in vitro and in vivo conditions against P. aphanidermatum and R. solani pathogens which cause rhizome rot and leaf blight diseases of turmeric respectively.

Materials and Methods

Sampling

Soil samples were collected from four different states of South India viz., Karnataka, Kerala, Tamilnadu and Andhra Pradesh. Samples were collected from top five cm of soil around healthy turmeric plants adhering to the roots. Collected soil samples (30 nos) were sealed in sterile polythene bags and transferred to ice box for transport. For endophyte isolation, healthy turmeric rhizomes (20 nos) were also collected from the above said regions in polythene bags, labeled and stored in refrigerator at 4°C in laboratory and processed within 48 h of collection.

Isolation of bacteria from rhizosphere

Ten gram of soil sample was placed in 95 ml sterile water (10−1) and shaken for 10 min. One (1.0) ml of this suspension was transferred into a 9 ml blank (10−2) and serially diluted up to 10−10. About 0.1 ml of each dilution from 10−8 to 10−10 series was added on Nutrient Agar (NA) medium and incubated at 37°C for 2–3 days. Morphologically distinct bacterial colonies were isolated and subcultured on NA medium, strains were temporarily cryopreserved at −20 °C in 40% glycerol for further studies.

Isolation of endophytic bacteria from rhizomes

The collected rhizomes were thoroughly washed in running tap water to remove soil particles adhered followed by dipping in phosphate buffer (per L:6.33 g of NaH2PO4; 16.5 g of Na2HPO4.7H2O; 200 ml Tween 40). Distilled water was used to remove foam of Tween 40. Rhizomes were further sterilized by sequential immersion in 70% ethanol for 2 min and in 3.5% sodium hypochloride for 3 min and then rinsed several times in sterile distilled water to remove surface sterilization agents. One gram of rhizome was ground in a sterile mortar and pestle with phosphate buffered saline (PBS) and the solution was made up to 10 ml. Serial dilutions from 10−1 to 10−4 were prepared and 0.1 ml of aliquots were spread onto NA medium amended with nystatin (50 mg ml−1; Sigma Aldrich, Bengaluru, India) in triplicates under laminar air flow to avoid external contamination and the plates were incubated for 7 days at 37°C. To verify the efficacy of surface sterilization of the rhizomes, 100 μl of the last rinse was added on NA medium and incubated. Morphologically distinct bacterial colonies were selected and pure cultures were preserved in 40% (v/v) glycerol solution at −20°C.

Pathogens

Virulent isolates of P. aphanidermatum (Accession No. KT315583) and R. solani (Accession No. KT366922) isolated from naturally infected turmeric rhizomes and leaves were obtained from the culture repository of the host Institute Department of Studies in Biotechnology, University of Mysore, Mysuru, India. Actively growing hyphae were successively transferred to the new PDA medium and the cultures were maintained on slants and stored at 4°C.

Characterization of rhizospheric and endophytic bacterial strains

The colony morphology, size, shape, colour and growth pattern of all the bacterial isolates was noted. Biochemical tests viz., Methyl red test, Voges-Proskauer test, Citrate test, Presence of oxidase and catalase, succinic acid, starch hydrolysis, ammonia production, casein hydrolysis were conducted to characterize the isolated bacterial strains (Cappuccino and Sherman, 1992). The Gram’s reaction was performed as per standard procedures (Holt et al., 1994). The motility of the bacteria was checked using hanging drop method and for the KOH solubility test, a loop full of bacterial strain was mixed with 3% KOH solution on a clean glass slide for 1 min and observed for formation of a thread like mass. The isolates were grouped based on the results of phenotypic and biochemical characteristics.

For molecular characterization, DNA extraction was done using HipurA Bacterial DNA Purifiation kit of Himedia. DNA was quantified by NanoDrop spectrophotometer (2000C, Thermo Scientific, Tokyo, Japan) and the quality was checked based on absorbance ratio 260/280. The integrity of the DNA was assayed by gel electrophoresis using 0.7% agarose gel. The DNA was amplified using universal primer pair of 16S rDNA, Forward 16S rDNA F 5′-CCAGACTCCTACGGGAGGCAGC-3′ and reverse 5′-GCTGACGAGAGCCATGCAGCACC-3′ (Sigma Aldrich, Bengaluru, India). The PCR reaction was performed in 50 μl final reaction volume containing 5 μl of 10X PCR buffer, 8 μl of 25 mM MgCl2, 2.5 μl of 1.25 mM dNTP, 0.2 μl of each primer (20 μM), 100 ng of DNA and 0.2 μl Taq DNA polymerase (5 U μl−1) (Sigma-Aldrich, Bengaluru, India) in a thermal cycler (Bio-Rad, CA, USA) programmed for initial denaturation at 94°C for 5 min, followed by 35 cycles of denaturation at 94°C for 45 s, primer annealing at 56°C for 45 s, extension at 72°C for 2 min. At the end of the amplification reaction, a final extension step was achieved at 72°C for 10 min. Ten microliters of the PCR products from each PCR reaction were electrophoresed on 1% agarose gel containing 5 mg ml−1 of ethidium bromide in a 1XTBE (PH 8.4) along with 100 bp molecular ladder (Sigma-Aldrich, Bengaluru, India) to estimate the size of the PCR products. The electrophoresis was carried out using 100 Volts. The gel was visualized and photographed using Gel Documentation system (Gel Doc 2000, Bio-Rad, CA, USA).

In vitro screening of rhizospheric and endophytic bacterial isolates for antagonism against P. aphanidermatum and R. solani

Antifungal activity in Dual culture method

All rhizospheric and endophytic bacterial isolates were screened for their antagonism in dual culture assays. The pathogen was inoculated in the middle of the petriplate containing PDA medium and bacteria were streaked 3 cm away on either sides of the pathogen and incubated at 28°C for 3 days. The petriplate inoculated with pathogen alone in the absence of antagonist served as control and the experiment was done in triplicates. The radial growth of fungal mycelium on each plate was measured and the percent inhibition of growth over control (absence of antagonists) was determined using the formula:

In the vicinity of bacterial colonies the morphology of hyphae of pathogens P. aphanidermatum and R. solani from PDA plates were observed under scanning electron microscope (SEM). The hyphal samples of pathogens were excised and fixed in 2.5% glutaraldehyde at 4°C for 2 h followed by washing in phosphate buffered saline (PBS) for 4 times, later dehydrated in a graded ethanol series (70%, 80%, 90%, and 100%) ten min each and air dried. It was then coated with gold in a POLARON, AU/PD sputter and scanned in SEM, S-3400N model (Hitachi, Tokyo, Japan) at 5.00 kV and the abnormalities in the fungal hyphae were recorded (Minaxi and Saxena, 2010).

Antifungal activity in liquid culture

Dual liquid culture method was used to test the antifungal activity in potato dextrose broth (PDB). Bacterial isolates that showed more than 70% antagonism in dual culture plates were selected (Table 1). 100ml of PDB was sterilized in 250 ml conical flask and inoculated with 5 × 5 mm disc of pathogenic fungal mycelia and1ml of bacterial culture (OD 0.25 at 590 nm). It was incubated at 28 ± 2°C for five days at 100 rpm. Dry weight of the fungal culture grown with bacterial strains and control (without bacterial strains) were recorded and differences were calculated according to Broekaert et al. (1990) and percent inhibition was calculated.

In in vitro study for antagonism only five rhizospheric and four endophytic bacteria showed more than 70% inhibition against pathogens (Table 1, 2). These isolates were tested for their plant growth promoting traits. For Indole acetic acid (IAA) production test, each isolate was inoculated to the sterile 15 ml Nutrient broth (NB) amended with L-tryptophan in test tubes and incubated at 28°C for 72 h in the dark (Gordon and Weber, 1951). Subsequently, 2 ml of this broth was centrifuged at 12,000 g for 10 min, followed by addition of 4 ml of Salkawaski reagent (Loper and Schroth, 1986) to the 1 ml of supernatant. The tubes were incubated at 37°C in the dark for 1 h. Development of a pink/red color in the medium indicated IAA production by the organisms.

Production of Hydrogen Cyanide (HCN) was determined in Nutrient agar (NA) supplemented with 4.4 g/l−1 of glycine (Lorck, 1948). The slant cultures were streaked on agar and Whatman No.1 filter paper strips dipped in 0.5% picric acid in 2% sodium carbonate solution were inserted from the top of each test tube, sealed with parafilm and incubated at 30°C for 4 days. A change of colour to brown or reddish-brown was recorded as positive (+) reaction.

Siderophore production of rhizospheric and endophytic bacteria was determined as described by Schwyn and Neilands (1987) using Chrome Azurol S (CAS) agar medium. The bacteria were spot inoculated and incubated at 30°C for 3–5 days. Development of yellow–orange halos around the colonies on CAS agar was considered as a positive result.

Phosphate solubalization ability of the strains was detected by spotting them on the Pikovskaya medium containing tricalcium phosphate and incubated at 28 ± 2°C for 2–3 days. Development of clear halo zone around the strains indicated positive result for phosphate solubalization (Pikovskaya, 1948).

Production of hydrolytic enzymes

Production of cell wall degrading enzymes such as protease and cellulase is a common mechanism used by bacteria to inhibit the growth of pathogenic microorganisms. For determining protease production one loop full of bacterial strains was streaked on skimmed milk agar plate (skimmed milk-100 g, peptone-5 g, agar-15 g and distilled water 1000 ml). After 48 h of incubation at 28°C, the development of clear zone around the streak was considered as a positive result. To determine celulolytic activity, carboxymethyl cellulose (CMC) was used in basal medium (NaNO3- 1 g, KCl- 1 g, K2HPO4- 1 g, MgSO4- 0.5 g, yeast extract- 0.5 g, agar-15 g, distilled water 1000 ml). The bacteria was streaked on the medium and incubated at 28°C for 3 days. The plates were flooded with 0.01% congo red solution for 15 min and destained using 1% NaCl solution for 5 min. A clear zone indicated the degradation of CMC and the bacteria was positive for cellulase production (Cappuccino and Sherman, 1992).

Evaluation of rhizospheric and endophytic bacteria for growth promotion and disease suppression in green house

our promising bacterial isolates two each from rhizopheric PGPR isolates viz., P. putida RBacDOB-S21, B. cereus RBacDOB-S24 and endophytic bacteria P. aeruginosa BacDOB-E19, Enterobacter sp. BacDOB-E21were selected for green house studies based on in vitro antagonism studies and PGP traits. Two sets of experiments were performed to analyze the efficacy of the bacterial isolates in controlling the rhizome rot and leaf blight diseases of turmeric under green house condition by using turmeric cultivar ‘Erode local’ (susceptible). Four replications were maintained for each treatment and each replication consisted of 5 earthen pots (20 cm diameter) in a completely randomized design (CRD) in a green house. The experiment was repeated twice. The talc-based formulation of the rhizospheric and endophytic bacterial isolates was prepared containing population densities of 3 × 108 bacteria/g talc powder (Shanmugam et al., 2011). The talc based formulations 20 g/L of each rhizospheric bacteria P. putida RBacDOB-S21, B. cereus RBacDOB-S24 and endophytic bacterial isolates P. aeruginosa BacDOB-E19, Enterobacteria sp. BacDOB-E21 were applied as rhizome treatment. The rhizomes were surface sterilized with 2% sodium hypochlorite for 1 min and soaked in sterile distilled water containing 20 g/l formulation. The suspension was drained off after 12 h and the rhizomes were air dried overnight under a sterile air stream. The rhizomes with three nodes were planted in earthen pots containing sterilized soil of 5 kg.

For first set of experiment, the pathogen P. aphanidermatum was multiplied on sand-corn meal medium and the rhizomes were infected after 30 days of planting at a ratio of 1:19 (sand-maize inoculum: soil), i.e., 300 g having 16 × 104 cfu g−1 of medium per pot (Shanmugam et al., 2013). For second set, the 30 day old BCA treated turmeric plants were challenge inoculated with R. solani by inserting young immature sclerotia, 2 sclerotia per sheath (Sriraj et al., 2014).

Soil applications (8 g) of biocontrol formulation containing 3 × 108 bacteria/g talc powder was applied three times upto 90 days at intervals of 15 days for first set of plants ( P. aphanidermatum inoculated). For the second set of plants (R. solani inoculated) soil application of bacteria was followed by foliar spray of rhizospheric and endophytic bacteria at 108 spores/ml suspended in water.

Carbendazim (0.1%) + Mancozeb (0.25%) combination was applied for rhizome treatment and soil drenching (20 ml) and for R. solani inoculated plants the rhizome and soil treatment was followed by spray of Carbendazim (0.1%) + Mancozeb (0.25%) served as fungicide control. The rhizomes without treatment and pathogens treated alone served as controls. The control plants showed systemic infection in 4–6 weeks of inoculation at temperature of 20–30°C. The disease severity on rhizome was assessed and the PDI was calculated as described below

PDI=Number of infected plantsTotal number of inoculated plants×100

The intensity of leaf blight disease was recorded after seven days of inoculation, with 0–9 scale of the Standard Evaluation System of rice, IRRI (2002) and expressed as percent disease index (Sriraj et al., 2014).

PDI=Sum of individual ratingsTotal number of plants observed×100Maximum grade

A separate set with four treatments along with untreated control and pathogenic control was maintained for rhizome colonization assay and growth promotion studies. The plant length and fresh rhizome yield of the plants were recorded at the time of harvest.

Rhizome colonization assay by Confocal Microscopy

BCA (PGPR B. cereus and P. aeruginosa) treated turmeric rhizomes (as explained earlier) of 60 days old, were removed intact from the soil. The rhizomes were thoroughly washed in running tap water followed by distilled water. The rhizomes were surface sterilized with 2% (w/v) sodium hypochoride solution for 30 s. Experiments were performed twice, and rhizomes from three plants were analyzed for each data. The rhizome material (1 cm) was transferred to trichloroacetic acid fixation solution (0.15% (wt/vol) trichloroacetic acid in 4:1 (vol/vol) ethanol/chloroform). Sections from rhizome were hand cut about 1 cm from the surface and approximately 50 μm thick segments were mounted on a microscope slide. Bacteria in rhizome segments were stained by 5 μl of Ethidium bromide (EtBr 1.25 mg ml−1) (Someya, 1995). Subsequently, segments were incubated at room temperature for 10 min. After incubation the segments were mounted on clean glass slides and examined immediately. Confocal fluorescence images were recorded on Advanced Spectral Confocal Microscope System-LSM 710 (Carl Zeiss, Jena, Germany). It was excited with a 514-nm laser line and detected at 552–693 nm, Channels EtBr and T-PMT were used (Hansen et al., 1997).

Statistical analysis

Statistical analyses were performed using Ans. SPSS, Version 17 (Chicago, IL, USA) and MS-Exel version 2007 (Microsoft, Washington, DC, USA). A completely randomized design was used for all the experiments, with 3 replications for each treatment. Differences between experimental outcomes were analysed using Tukey’s HSD test and P ≤ 0.05 was considered not significantly different.

Results

Morphological and Biochemical traits of rhizospheric and endophytic bacteria isolated from turmeric

A total of 30 PGPR isolates from Rhizosphere and 20 endophytic bacteria from different geographic regions of South India viz., Karnataka, Kerala, Tamilnadu and Andhra Pradesh states were obtained. The isolates belonged to Bacillus, Pseudomonas, Arthrobacter, Enterobacter, Alcaligenes, Acinetobacter, Ochrobactrum, Exiquebacterium, Rhizobium, Klebsiella, Citrobacter and Terribacillus species. All the isolates were motile rods. Out of 50 isolates, 43 were Gramnegative while 7 were Gram-positive. Besides, 12 for methyl red test, 42 for citrate utilization, 33 for oxidase, 4 for VP, 22 for succinic acid production, 1 for starch hydrolysis, 31 isolates for NH3 production, 41 isolates for casein hydrolysis were found positive, while all the isolates were positive for catalase production (Supplementary Table 1).

Molecular characterization of rhizospheric and endophytic bacteria

The quality of genomic DNA of rhizospheric and endophytic bacteria was good as evident from the ratio of 260/280, which was 1.72. All the bacterial isolates the DNA was subjected to PCR amplification with specific primer for the 16s rDNA region which generated bands ranging from 630–700 bp. The sequences of 30 rhizospheric and 20 endophytic bacterial isolates showed 97–99% similarity with the species in Genebank during Blast analysis. The Blast search confirmed the presence of Bacillus, Pseudomonas, Arthrobacter, Enterobacter, Alcaligenes, Acinetobacter, Ochrobactrum, Exiquebacterium, Rhizobium, Klebsiella, Citrobacter and Terribacillus species. All the 50 bacterial sequences were submitted to Genebank (NCBI) and their accession numbers were obtained (Table 1, 2).

The Phylogenetic trees of rhizospheric and endophytic bacterial isolates constructed from 16s rDNA sequences along with the related reference species retrieved from Genbank of NCBI confirmed these isolates belong to Bacillus, Pseudomonas, Arthrobacter, Enterobacter, Alcaligenes, Acinetobacter, Ochrobactrum, Exiquebacterium, Rhizobium, Klebsiella, Citrobacter and Terribacillus species by clustering of each of the isolate to its corresponding group (Fig. 1, 2).

In vitro antagonism

All the isolates were screened against P. aphanidermatum and R. solani by dual culture method (Fig. 1, 2). Five PGPR isolates viz., RBacDOB-S4, RBacDOB-S16, RBacDOB-S21, RBacDOB-S24, RBacDOB-S70 out of 30 rhizosoheric bacterial isolates and four endophytic bacteria BacDOB-E12, BacDOB-E15, BacDOB-E19, BacDOB-E21 out of 20 exhibited > 70% growth inhibition against both the pathogens (Table 1). SEM studies showed that the endophytes cause deformities in the mycelia of both P. aphanidermatum and R. solani pathogens. The deformities included hyphal fragmentation, perforation, desiccation of hyphae and mycelia degeneration (Fig. 3, 4) which finally resulted in fungal death.

Rhizome colonization assay by Confocal Microscopy

The colonization in turmeric rhizomes was analysed by confocal microscopy. The results revealed that the PGPR B. cereus RBacDOB-S24 and endophyte P. aeruginosaBac-DOB-E19 treated rhizomes showed colonization between the cells (Fig. 8).

Discussion

In this study, 30 PGPR were isolated from turmeric rhizosphere and 20 endophytes from healthy rhizome. The identity of the isolates was confirmed by morphological, biochemical and 16S rDNA sequences. The 16S rDNA sequences were submitted to Genbank (NCBI) (Table 1). A majority of the isolates belonged to Bacillus, Pseudomonas, Arthrobacter, Enterobacter, Alcaligenes, Acinetobacter, Ochrobactrum, Exiquebacterium, Rhizobium, Klebsiella, Citrobacter and Terribacillus species. All the isolates (30 PGPR and 20 endophytes) were screened for nutrient solubilization, biochemical traits and antagonism in order to select the isolates that showed the most promising results with regard to growth promotion and biocontrol of rhizome rot and leaf blight diseases in turmeric plants.

Many of the soil-borne fungal diseases have been successfully controlled by the use of antagonists (Weller, 1988). The in vitro screening of rhizosphere bacterial isolates and endophytic bacteria for antagonism against P. aphanidermatum an R. solani indicated that nine isolates exhibited > 70% inhibition (Table 1, 2) of both the pathogens in dual culture and liquid culture assays. Endophytic bacteria, used as whole cells (Rajendran and Samiyappan, 2008) and cell-free culture filtrates (Li et al., 2012) suppressed some plant pathogenic fungi due to antimicrobial compounds that cause alteration in structural architect and lysis of mycelia (Yuan et al., 2012). Our SEM results also revealed the morphological deformities of mycelia of both the pathogens. Similar observations have been reported in Pythium myriotylum due to the effect of extracellular metabolites by Bacillus sp. (Jimtha et al., 2016).

The rhizospheric and endophytic isolates have also exhibited significant plant growth promoting traits. Bacteria producing IAA promotes plant growth directly by increasing root surface area and length by stimulating plant cell elongation or by affecting the cell division thereby providing greater access to soil nutrients by plants (Glick, 1995). In plant growth promotion analysis endophytic strains P. aeruginosa BacDOB-E19 and PGPR strains B. cereus RbacDOB-S24 produced significant amount of IAA, earlier reported in B. cereus (Rana et al., 2011) and P. putida (Jasim et al., 2014). Production of siderophores, indirectly influence the plant growth by binding to the available form of iron in the rhizosphere making it unavailable to the phytopathogens and protecting the plant health. Siderophore production by Bacillus sp. and Pseudomonas sp. in this study evidenced for one of the biocontrol mechanism similar to previous reports (Jasim et al., 2014; Kumar et al., 2016). Siderophore produced by Pseudomonas sp. has been reported to be an important mechanism of biological control of Pythium diseases (Matthijs et al., 2007). The results supported that endophytic and PGPR bacterial strains viz., B. cereus, P. aeruginosa, P. putida solubilize phosphate as reported previously for Bacillus sp. and P. putida (Forchetti et al., 2007; Pandey et al., 2006). In the present study, B. cereus and P. aeruginosa strains showed production of HCN similar to the previous results reported for Pseudomonas strains that controlled the plant root pathogens including F. oxysporum and R. solani by production of siderophores, HCN and lytic enzymes (Nagrajkumar et al., 2004). The selected strains B. cereus and P. aeruginosa produced cell wall degrading enzymes such as cellulases and proteases, which reported earlier as important in breakdown of cell walls of oomycete pathogens such as Phytopthora (Valois et al., 1996) and Pythium spp. (El-Tarabily et al., 2009). Similarly, the lytic enzyme production by rhizospheric P. fluorescence are known to be involved in the control of pathogens like F. oxysporum and R. solani (Nagrajkumar et al., 2004).

The four promising biocontrol agents (BCA’s), two from rhizosphere and two from endophytes were then tested in the green house for their disease suppression and plant growth promotion abilities compared to untreated and pathogenic controls. Green house results suggested that the PGPR PGPR B. cereus and endophyte P. aeruginosa showed significant disease reduction also enhanced the yield of turmeric when compared to untreated control. Similar to our reports on turmeric, there are several studies on growth promotion by PGPR in other crops like maize (Egamberdiyeva, 2007), tomato (Almaghrabi et al., 2013), common bean (Martins et al., 2013) and ginger (Dinesh et al., 2015) have been reported. Endophyte B. cereus and P. fluorescens possesses biocontrol potential in crops like cotton and chilli, against root rot and damping off caused by R. solani and P. aphanidermatum respectively (Muthukumar et al., 2011; Pleban et al., 1997). Bacterial endophytes viz., B. cereus, B. thuringiensis, B. pumilis, P. putida and Clavibacter michiganensis, isolated from turmeric rhizomes exhibited PGP traits and antifungal activity against F. solani, A. pullulans, Alternaria alternata and B. fulva pathogens (Kumar et al., 2016). The culture supernatant of B. cereus QQ308 was active against numerous plant pathogenic fungi and has used in biological control (Chang et al., 2007). The potential of Bacillus cereus as a biocontrol agent against Fusarium solani causing rhizome rot in turmeric has been reported previously (Chauhan et al., 2016). Control of anthracnose rot caused by Colletotrichum acutatum in harvested loquat fruit inducing disease by biocontrol agent B. cereus was reported (Wang et al., 2014). The endophytes viz., P. aeruginosa, P. putida and B. megaterium associated with black pepper were reported as effective antagonists for biological control of Phytophthora foot rot which recorded over 70% disease suppression in green house trials (Aravind et al., 2009).

The PGPR isolated from rhizosphere of ginger viz., B. amyloliquefaciens and S. marcescens markedly reduced the soft rot incidence of ginger rhizome caused by P. myriotylum and showed marked increase in rhizome yield compared to chemical treatments (Dinesh et al., 2015). The potent PGPR strains should fulfill at least two of the criteria such as colonization, plant growth stimulation and biocontrol (Beneduzi et al., 2012). Nevertheless, in present study PGPR B. cereus and endophyte P. aeruginosa both possess direct PGP activities like IAA production and phosphate solubilization and indirect PGP activities like antifungal activity, siderophore, HCN production and produce lytic enzymes protease and cellulase. In vivo evidence suggest that PGPR B. cereus RBacDOB-S24 and endophyte P. aeruginosa BacDOB-E19 suppressed the disease incidence of rhizome rot and leaf blight significantly and expressed high yield. Hence, these strains can be explored as potential biocontrol agents in order to control the rhizome rot and leaf blight diseases in turmeric which helps to reform the chemical fungicide based disease management approaches.

The present study revealed the importance of isolating, screening of bacteria for multiple PGP and biocontrol traits through greenhouse experiments in turmeric. In this study, based on in vitro experiments, two strains viz., PGPR B. cereus RBacDOB-S24 and endophyte P. aeruginosa Bac-DOB-E19 (Out of the 30 PGPR and 20 endophytes from turmeric) exhibited multiple plant growth promoting traits. The results of green house evidenced these strains suppressed the disease incidence of rhizome rot and leaf blight significantly, and markedly enhanced the yield in turmeric compared to untreated control and chemical treatments like Carbendazim- mancozeb. Also, B. cereus RBacDOB-S24 and endophyte P. aeruginosa BacDOB-E19 treated rhizomes showed colonization in the cells. The study confirms the potential of PGPR B. cereus RBacDOB-S24 and endophyte P. aeruginosa BacDOB-E19 as biocontrol agents (BCA’s) for sustainable turmeric cultivation. For the best of our knowledge, this is the first report on the strains PGPR B. cereus RBacDOB-S24 and endophyte P. aeruginosa BacDOB-E19 as biocontrol agents (BCA’s) against P. aphanidermatum and R. solani pathogens of turmeric. Further studies concerning field applications and stable bioformulations are in progress.

Acknowledgments

This work was carried out with the financial assistance from the Department of Science and Technology (DST), Government of India, New Dehli, under the Women Scientist Scheme (DST-WOS A) awarded to Mrs. Vinaya Rani. G (DST sanction No.SR/WOS-A/LS-104/2013 (G) dated 22.04.2014. The authors extend thanks to Dr. K. Ramachandra Kini, Associate Professor, Department of Biotechnology, University of Mysore, Mysore for his help in Phylogenetic analysis of endophytes. We also thank Institution of Excellence (IOE) at University of Mysore for providing instrumentation facility.

Fig. 5. Antagonistic activity of rhizospheric bacterial isolates against P. aphanidermatum and R. solani at two different time intervals in dual liquid culture assay. Py1 and Rh1 = % inhibition of fungi after 48 h; Py2 and Rh2 = % inhibition of fungi after 120 h. Each value is the mean for four replicates (n = 4) and bars sharing the same letters are not significantly different (P ≤ 0.05) according to Turkey’s HSD. The vertical bars indicates the standard error.

Fig. 6. Antagonistic activity of endophytic bacterial isolates against P. aphanidermatum and R. solani at two different time intervals in dual liquid culture assay. Py1 and Rh1 = % inhibition of fungi after 48 h; Py2 and Rh2 = % inhibition of fungi after 120 h. Each value is the mean for four replicates (n = 4) and bars sharing the same letters are not significantly different (P ≤ 0.05) according to Turkey’s HSD. The vertical bars indicates the standard error.

Molecular identification of rhizospheric bacteria isolated from turmeric using 16S rDNA sequences and their antagonistic effect on the pathogenic fungus Pythium aphanidermatum and Rhizoctonia solani

Sl No

Geographical location (GPS)

Variety of turmeric

Rhizospheric bacterial isolate (PGPR)

Closest related species

% Identity

Accession No.

% Growth inhibition of P. aphanidermatum

% Growth Inhibition of R. solani

1

Chamaraja nagar, Karnataka12.0526° N, 77.2865° E

BSR 2

RBacDOB-S1

Pseudomonas plecoglossicida

98

KY818291

65.0±0.79h

63.6±0.77i

2

Kollegal, Karnataka12.1537° N, 77.1111° E

BSR 2

RBacDOB-S4

Pseudomonas plecoglossicida

97

KY818292

74.6±0.57b

72.2±0.65c

3

Dharwad, Karnataka15.4589° N, 75.0078° E

Local

RbacDOB-S6

Stenotrophomonas sp.

99

KY883574

71.8±0.37d

65.2±0.77h

4

Dharwad, Karnataka15.4589° N, 75.0078° E

Local

RBacDOB-S9

Stenotrophomonas maltophilia

97

KY883576

67.6±0.33f

67.9±0.57f

5

Dakshina Kannada, Karnataka12.8438° N, 75.2479° E

Local

RbacDOB-S10

Pseudomonas monteilii

99

KY883577

67.3±0.5f

67.5±0.33f

6

Dakshina Kannada, Karnataka12.8438° N, 75.2479° E

Local

RbacDOB-S11

Pseudomonas aeruginosa

97

KY883578

58.9±0.79l

64.2±0.75h

7

Erode, Tamilnadu11.3410° N, 77.7172° E

Erode Local

RBacDOB-S14

Pseudomonas aeruginosa

99

KY883580

56.2±0.56n

53.6±0.33o

8

Chamarajanagar, Karnataka12.0526° N, 77.2865° E

BSR 1

RbacDOB-S16

Stenotrophomonas maltophilia

98

KY883582

73.7±0.73c

71.4±0.37d

9

Chamarajanagar, Karnataka12.0526° N, 77.2865° E

BSR 1

RBacDOB-18

Brevibacillus agri

98

KY883583

57.0±0.32m

59.0±0.54l

10

Hassan, Karnataka13.0068° N, 76.0996° E

Local

RbacDOB-S20

Pseudomonas hibiscicola

97

KY883584

73.2±0.57c

57.6±0.73m

11

Bavanisagar, Tamilnadu11.4792° N, 77.1341° E

BSR 2

RbacDOB-S21

Pseudomonas putida

98

KY883585

79.8±0.97a

74.6±0.59b

12

Dharwad, Karnataka15.4589° N, 75.0078° E

Local

RbacDOB-S23

Pseudomonas aeruginosa

98

KY883586

59.3±0.57l

65.0±0.67h

13

Gobi, Tamilnadu11.4504° N, 77.4300° E

BSR 2

RbacDOB-S24

Bacillus cereus

99

KY883587

79.8 ±0.37a

76.6±0.87a

14

Coimbatore, Tamilnadu11.0168° N, 76.9558° E

BSR 2

RBacDOB-S26

Bacillus thuringiensis

99

KY883598

66.0±0.33g

66.2±0.33g

15

Coimbatore, Tamilnadu11.0168° N, 76.9558° E

BSR 2

RbacDOB-S29

Pseudomonas alcaliphila

98

KY883588

65.0±0.67h

69.5±0.57e

16

Dharwad, Karnataka15.4589° N, 75.0078° E

Local

RbacDOB-S30

Pseudomonas putida

97

KY883589

60.2±0.77k

60.0±0.81k

17

Kollegal, Karnataka12.1537° N, 77.1111° E

Salem local

RbacDOB-S35

Bacillus megaterium

99

KY883590

57.4±0.61m

59.0±0.57l

18

Calicut, Kerala11.2588° N, 75.7804° E

Alleppey Finger

RbacDOB-S36

Stenotrophomonas maltophilia

97

KY883591

61.1±0.57j

61.4±0.44j

19

Calicut, Kerala11.2588° N, 75.7804° E

Alleppey Finger

RbacDOB-S40

Stenotrophomonas maltophilia

97

KY883592

58.8±0.77l

61.0±0.57j

20

Salem, Tamilnadu11.6643° N, 78.1460° E

Salem local

RbacDOB-S41

Pseudomonas plecoglossicida

98

KY883593

61.8±0.59j

64.2±0.33h

21

Mysore, Karnataka12.2958° N, 76.6394° E

Local

RbacDOB-S51

Pseudomonas stutzeri

98

KY883594

63.4±0.75i

60.0±0.57k

22

H.D.kote, Karnataka12.0879° N, 76.331° E

Local

RbacDOB-S52

Ochrobactrum sp.

98

KY883595

56.0±0.67n

58.2±0.37l

23

Guntur, Andrapradesh16.3067° N, 80.4365° E

Duggirala

RbacDOB-S53

Pseudomonas aeruginosa

97

KY883596

66.7±0.66g

68.9±0.97e

24

Hassan, Karnataka13.0068° N, 76.0996° E

Local

RbacDOB-S56

Bacillus cereus

98

KY883597

56.0±0.57n

57.6±0.91m

25

H.D.Kote, Karnataka12.0879° N, 76.331° E

Local

RBacDOB-S57

Exiquebacterium aurantiacum

99

KY924598

50.1±0.77q

53.0±0.87o

26

Wayanad, Kerala11.6854° N, 76.1320° E

Alleppey Finger

RBacDOB-S62

Acinetobacter sp.

97

KY971450

50.1±0.57q

52.6±0.81o

27

Kadapa, Andhra Pradesh14.4674° N, 78.8241° E

Tekurpeta

RBac-DOBS70

Enterobacter sp.

99

KY971459

70.1±0.73e

69.0±0.57e

28

Kadapa, Andhra Pradesh14.4674° N, 78.8241° E

Sugandham

RbacDOB-S72

Rhizobium pusense

98

KY883605

63.1±0.57i

61.8±0.53j

29

Calicut, Kerala11.2588° N, 75.7804° E

Alleppey Finger

RBacDOB-S74

Brevibacillus brostelensis

99

KY982872

54.6±0.63o

54.6±0.97n

30

Salem, Tamilnadu11.6643° N, 78.1460° E

Salem local

RBacDOB-S78

Alcaligenes faecalis

99

KY982875

53.0±0.97p

57.0±0.85m

Values are the mean of three independent replicates (n = 3). ± indicate standard errors. Mean followed by the same letter (s) within the same column are not significantly (P ≤ 0.05) different according to Tukey’s HSD.

Table 2

Molecular identification of endophytic bacteria isolated from turmeric rhizome using 16S rDNA region and their antagonistic effect on the pathogenic fungus P. aphanidermatum and R. solani

Sl No.

Geographical location (GPS)

Variety of turmeric

Endophytic bacterial isolate

Closest related sps.

% Identity

Accession No.

% Growth inhibition of P. aphanider matum

% Growth inhibition of R. solani

1

Chamaraja nagar, Karnataka12.0526° N, 77.2865° E

BSR 2

BacDOB-E2

Alcaligenes faecalis

97

KY883599

64.0±0.57g

62.6±0.67i

2

Hassan, Karnataka13.0068° N, 76.0996° E

Local

BacDOB-E3

Pseudomonas sp.

99

KY883600

61.6±0.44h

65.2±0.33h

3

Kollegal, Karnataka12.1537° N, 77.1111° E

BSR 2

BacDOB-E4

Pseudomonas aeruginosa

99

KY883601

69.0±0.77e

67.0±0.57g

4

Dharwad, Karnataka15.4589° N, 75.0078° E

Local

BacDOB-E5

Citrobacter sp.

99

KY883602

70.8±0.57d

68.2±0.43f

5

H.D Kote Karnataka12.0879° N, 76.331° E

Local

BacDOB-E7

Terribacillus saccharophilus

98

KY883603

57.0±0.63k

59.4±0.57l

6

Mysore, Karnataka12.2958° N, 76.6394° E

Local

BacDOB-E8

Pseudomonas aeruginosa

99

KY883604

69.6±0.49e

68.9±0.33f

7

Dandeli, Karnataka15.2497° N, 74.6174° E

Local

BacDOB-E9

Pseudomonas plecoglossicida

97

KY883606

66.3±0.57f

69.5±0.37e

8

Madikeri, Karnataka12.4244° N, 75.7382° E

Local

BacDOB-E11

Pseudomonas aeruginosa

99

KY883607

58.9±0.63i

60.2±0.57k

9

Kollegal, Karnataka12.1537° N, 77.1111° E

BSR 1

BacDOB-E12

Acinetobacter sp.

98

KY924605

73.2±0.73c

71.6±0.33d

10

Coimbatore, Tamilnadu11.0168° N, 76.9558° E

BSR 2

BacDOB-E14

Alcaligenes sp.

99

KY776473

66.8±0.51e

65.8±0.57h

11

Salem, Tamilnadu11.6643° N, 78.1460° E

Salem local

BacDOB-E15

Pseudomonas aeruginosa

99

KY883608

73.8±0.61c

72.6±0.77c

12

Gobi, Tamilnadu11.4504° N, 77.4300° E

Erode local

BacDOB-E17

Pseudomonas aeruginosa

99

KY924595

58.0±0.33j

60.0±0.79k

13

Bavanisagar, Tamilnadu11.4792° N, 77.1341° E

BSR 2

BacDOB-E18

Arthrobacter sp.

97

KY924596

73.2±0.43c

66.6±0.74g

14

Erode, Tamilnadu11.3410° N, 77.7172° E

BSR 1

BacDOB-E19

Pseudomonas aeruginosa

99

KY924597

76.9±0.57a

74.6±0.53a

15

Sathyamangalam, Tamilnadu11.5048° N, 77.2384° E

BSR 2

BacDOB-E20

Bacillus cereus

99

KY924599

59.3±0.63i

63.0±0.57i

16

Guntur, Andrapradesh16.3067° N, 80.4365° E

Duggirala

BacDOB-E21

Enterobacter sp.

98

KY924600

75.7 ±0.57b

73.4±0.87b

17

Kadapa, Andrapradesh14.4674° N, 78.8241° E

Sugandham

BacDOB-E22

Bacillus cereus

98

KY924601

66.0±0.83e

68.2±0.47f

18

Wayanad, Kerala11.6854° N, 76.1320° E

Alleppey Finger

BacDOB-E34

Acinetobacter sp.

97

KY924606

57.0±0.47k

54.5±0.53n

19

Wayanad, Kerala11.6854° N, 76.1320° E

Alleppey Finger

BacDOB-E47

Enterobacter sp.

98

KY924602

61.2±0.77h

62.0±0.59j

20

Calicut, Kerala11.2588° N, 75.7804° E

Alleppey Finger

BacDOB-E52

Klebsiella sp.

99

KY986971

57.2±0.33k

58.6±0.77m

Values are the mean of three independent replicates (n = 3). ± indicate standard errors. Mean followed by the same letter (s) within the same column are not significantly (P ≤ 0.05) different according to Tukey’s HSD.